Immune cells, those responsible for fending off a foreign substance like an invading virus, are found to activate like the flicking of a light switch from off to on, or rather more like the striking of a match to produce a flame. New research from the Stowers Institute for Medical Research in the lab of Associate Investigator Randal Halfmann, PhD, has revealed that the mechanisms involved for invoking an immune system response are inherently connected to the thermodynamic potential for a specific protein complex to assemble when a pathogen is encountered.

For a small change in energy, or an infinitesimally small stimulus—like the binding of just one molecule of virus genome by immune cell proteins—to produce a very large, irreversible response requires a pre-paid energy storage scheme within each immune cell. The findings have broad implications for not only uncovering the causes and progression of inflammatory illness in humans but also to understand other age-related diseases that arise from proteins that can surmount energy barriers to self-assemble, or nucleate, into structural complexes involved in devastating diseases like Alzheimer’s.

At the cellular level, to create order from disorder necessitates particular conditions that allow proteins capable of self-assembly to reach high concentrations. In essence, this protein supersaturation increases the sensitivity of invoking a response as only a tiny fraction of the protein molecules need to respond directly to the stimulus. “Protein self-assembly involves a disorder-to-order phase transition,” said Halfmann. “This imposes an energy barrier that allows the immune cell proteins to exist at the high concentrations required to drive a rapid and sensitive cell-wide response to stimulation.”

In response to recognition of an invading molecule, a large protein complex called a signalosome is assembled which stimulates a signaling pathway that activates our immune system. In a recent study published in eLife on June 21, 2022, Predoctoral Researcher Alejandro Rodríguez Gama, from the Halfmann Lab, revealed that polymerization—the assembly of a large structure from smaller components—of the immune cell specific signalosome, CBM, activates immune cells in a binary, “all-or-nothing” fashion. They also resolved the assembly mechanism of the CBM signalosome and discovered that a component of CBM, the adaptor protein BCL10, intrinsically encodes the switch in the form of an energy barrier to polymerization.

Most vertebrates, including humans, have two different immune system responses that act to confer protection to danger. Innate immunity, or the immune system a species is born with, includes both physical barriers, like skin and mucus, along with specialized cells that can engulf or produce enzymes that destroy potential invaders. This first line of defense is accompanied by adaptive immunity, the response a species acquires from previous pathogen exposure; this leads to the production and proliferation of white blood cells that recognize, and remember, foreign molecules with remarkable specificity.

A fundamental quest in structural biology is to understand how the specific sequence of amino acids, the building blocks of proteins, enables a protein to fold into a structure associated with a particular function. While some scientists have focused on resolving the structures of various signalosomes, the motivation behind this study was to identify why, exactly, the elegant, elaborately ordered structure of CBM underlies the catastrophic event of an immune system response.

Gama and coauthors investigated how CBM signalosome assembly activates a particular transcription factor—a protein that controls transcription of RNA from DNA by binding to a specific sequence of DNA—called NF-kB that plays a central regulating role in both innate and adaptive immune response to infection. Mutations in the proteins that compose CBM can result in problems regulating NF-kB activity with serious implications like cancer, inflammatory, and autoimmune diseases.

The researchers found that a large energy barrier prevents signalosome assembly from easily occurring but that this same barrier enables BCL10 to naturally be supersaturated, or reach very high, metastable concentrations within a cell. This in turn increases the certainty and rapidity of assembly at some point in the cell’s future.

“Imagine a dam holding water back from flowing down a river—that dam is the nucleation barrier,” said Halfmann. “As water is rising behind the dam, energy is accumulating. If you punch a hole at any location, the consequence is the same.”

The energy barrier for nucleation is encoded in the sequence of the BCL10 protein; this along with the protein’s relative overexpression primes it to initiate CBM self-assembly upon the slightest stimulation, like the striking of a match.

“Every possible ordered conformation is another leak in the dam,” said Halfmann. “Thus, the way a sequence encodes a nucleation barrier is that the sequence has evolved to prevent all ordered conformations except for one.”

The collective findings indicate the discovery of a novel paradigm where form meets function. The thermodynamic improbability of nucleation serves to fine tune our immune system to activate under precise, all-or-none conditions; however, the energy barrier that encourages protein supersaturation and the probability that nucleation will eventually occur indicates that inflammation in progressive and age-related illness is inevitable.

“The key regulator of inflammation turns out to be a kinetic barrier that holds back protein aggregation, and this imposes a theoretical limit on lifespan,” said Halfmann. “Basically, we’re trading longevity for an immune system, or the greater certainty of life right now at the expense of potential longer life.”

It's not all bad news though. What makes this work so essential is that it elucidates specific thermodynamic forces that may be responsible for chronic and age-related inflammation. In the future, it may be possible to develop therapeutic solutions to modify our cell’s natural nucleation barriers to prevent, or at the very least to delay, inflammatory disease in humans.

Coauthors include Tayla Miller, Jeffrey J. Lange, PhD, and Jay Unruh, PhD.

This work was funded by the National Institute of General Medical Sciences (award R01GM130927) and the National Institute on Aging (award F99AG068511) of the National Institutes of Health, the American Cancer Society (award RSG-19-217-01-CCG), and by institutional support from the Stowers Institute for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.